Conventionally, methods of multiple microorganism detection using multiplex PCR are well known. For example the following methods have been reported: a method of multiple detection of bacteria of pathogenic Escherichia coli O157, Salmonella spp. and Listeria monocytogenes, targeting vegetables and fruits (for example, see non-patent document 1); a method of multiple detection of pathogenic Escherichia coli O157 and Salmonella spp. in foods (for example, see non-patent document 2); a method of multiple detection of various bacteria of pathogenic Escherichia coli O157, Salmonella spp., Listeria monocytogenes and Campylobacter spp., targeting milk (for example, see non-patent document 3); a method for multiple detection of Salmonella spp. and Listeria monocytogenes in foods (for example, see non-patent document 4); a method for multiple detection of Escherichia coli including pathogenic Escherichia coli O157, in foods (for example, see non-patent document 5); and a method for multiple detection of bacteria of pathogenic Escherichia coli O157, Salmonella spp. and Listeria monocytogenes, targeting milk (for example, see non-patent document 6). Further, primers for detecting Escherichia coli (for example, see patent document 1) and primers for detecting O-antigen of pathogenic Escherichia coli O157 (for example, see patent document 2) are known as primers for multiplex PCR.
On the other hand, as for methods for extracting DNAs of microorganisms, the following methods are known: a method using lytic enzyme Achromopeptidase and the like to mycobacterium such as tubercle bacilli (for example, see patent document 3); a method using lytic enzyme Achromopeptidase and the like to Gram negative bacteria and Gram positive bacteria (for example, see patent document 4); a method using proteinase K or Achromopeptidase and the like to Legionella (for example, see patent document 5); and a method using protein denaturant, reducing agent, surfactant, chelator and the like to Escherichia coli and the like (for example, see patent document 6).    Patent document 1: Japanese Laid-Open Patent Application No. 2001-95576    Patent document 2: Japanese Laid-Open Patent Application No. 11-332599    Patent document 3: Japanese Laid-Open Patent Application No. 6-165676    Patent document 4: Japanese Laid-Open Patent Application No. 9-500793    Patent document 5: Japanese Laid-Open Patent Application No. 5-317033    Patent document 6: Japanese Laid-Open Patent Application No. 6-289016    Non-patent document 1: Japanese Journal of Food Microbiology, Vol. 19, No. 2, 47-55, 2002    Non-patent document 2: Journal of Industrial Microbiology & Biotechnology, 21, 92-98, 1998    Non-patent document 3: Milchwissenschaft, 55(9), 500-503,    Non-patent document 4: Journal of Food Protection, Vol. 64, No. 11, 1744-1750, 2001    Non-patent document 5: Molecular and Cellular Probes, 13, 291-302, 1999    Non-patent document 6: Food Microbiology, 20, 345-350, 2003